The hepatic synthesis of oxalate makes an important contribution to the urinary excretion of oxalate and the formation of calcium oxalate kidney stones. Inherited deficiencies of key enzymes in the oxalate biosynthetic pathway result in a greatly increased oxalate synthesis and a cascade of pathological events that can culminate with death at a young age. The long-term objective of the research in this proposal are to elucidate the steps in the biosynthetic pathway, to determine how the pathway is regulated, to understand the physiological significance of the pathway, and to use this knowledge to develop effective therapies to decrease its synthesis. Hep G2 cells are an established line of cells derived from a human hepatoma and are known to retain many important liver functions, including the expression of specific liver enzymes involved in oxalate synthesis. These cells have been shown to synthesize and excrete oxalate into the growth medium and will be used as an experimental system to study oxalate synthesis. The specific aims of this proposal are (a) to determine the levels of intracellular metabolites involved in oxalate synthesis, (b) to identify biosynthetic pathways by including oxalate precursors in the growth medium, (c) to examine the effects of metabolic and sex hormones on intracellular metabolite levels and oxalate synthesis and thereby identify regulatory steps, and (d) to study inhibitors of oxalate synthesis to verify their mode of action and assess their use as pharmacological agents to decrease oxalate synthesis. The experimental design will consist of growing cells to confluency in a serum-containing medium and then exposing the cells to a serum-free test medium. Oxalate synthesis will be measured by a specific enzymatic procedure using the enzyme, oxalate oxidase. Intracellular metabolites will be measured by high performance liquid chromatography. The proposed experiments should lead to a greatly increased understanding of oxalate synthesis, its regulation, and approaches that can be used to decrease its synthesis. This knowledge should prove valuable in the treatment of the primary hyperoxalurias and calcium oxalate nephrolithiasis.